Ischemic or traumatic injuries to the brain or spinal cord often produce irreversible damage to central nervous system (CNS) neurons and to their processes. These injuries are major problems to society as they occur frequently, the damage is often severe, and at present there are still no effective treatments for acute CNS injuries. Clinically, ischemic cerebral stroke or spinal cord injuries manifest themselves as acute deteriorations in neurological capacity ranging from small focal defects-, to catastrophic global dysfunction-, to death. It is currently felt that the final magnitude of the deficit is dictated by the nature and extent of the primary physical insult, and by a time-dependent sequence of evolving secondary phenomena which cause further neuronal death. Thus, there exists a theoretical time-window, of uncertain duration, in which a timely intervention might interrupt the events causing delayed neurotoxicity. However, little is known about the cellular mechanisms triggering and maintaining the processes of ischemic or traumatic neuronal death, making it difficult to devise practical preventative strategies. consequently, there are currently no clinically useful treatments for cerebral stroke or spinal cord injury.
In vivo, a local reduction in CNS tissue perfusion mediates neuronal death in both hypoxic and traumatic CNS injuries. Local hypoperfusion is usually caused by a physical disruption of the local vasculature, vessel thrombosis, vasospasm, or luminal occlusion by an embolic mass. Regardless of its etiology, the resulting ischemia is believed to damage susceptible neurons by impacting adversely on a variety cellular homeostatic mechanisms. Although the nature of the exact disturbances is poorly understood, a feature common to many experimental models of neuronal injury is a rise in free intracellular calcium concentration ([Ca.sup.2+ ].sub.i). Neurons possess multiple mechanisms to confine [Ca.sup.2+ ].sub.i to the low levels (about 100 nM)-necessary for physiological function. It is widely believed that a prolonged, rise in [Ca.sup.2+ ].sub.i deregulates tightly-controlled Ca.sup.2+ -dependent processes, causing them to yield excessive reaction products, to activate normally quiescent enzymatic pathways, or to inactivate regulatory cytoprotective mechanisms. This, in-turn, results in the creation of experimentally observable measures of cell destruction such as lipolysis, proteolysis, cytoskeletal breakdown, pH alterations, and free radical formation.
The classical approach to preventing Ca.sup.2+ neurotoxicity has been through pharmacological blockade of Ca.sup.2+ entry through Ca.sup.2+ channels and/or of excitatory amino acid (EAA)-gated channels. Variations on this strategy often lessen EAA-induced or anoxic cell death in vitro, lending credence to the Ca.sup.2+ -neurotoxicity hypothesis. However, a variety of Ca.sup.2+ channel- and EAA-antagonists fail to protect against neuronal injury in vivo, particularly in experimental Spinal Cord Injury (SCI), head injury, and global cerebral ischemia. It is unknown whether this is due to insufficient drug concentrations, inappropriate Ca.sup.2+ influx blockade, or to a contribution from non-Ca.sup.2+ dependent neurotoxic processes. It is likely that Ca.sup.2+ neurotoxicity is triggered through different pathways in different CNS neuron types. Hence, successful Ca.sup.2+ -blockade would require a polypharmaceutical approach.
It is well-known that calcium buffer salts and their acetoxymethyl esters have been used extensively to study various aspects of cellular neurophysiology. These studies have focused primarily on experiments involving isolated tissue preparations in vitro.
Kudo et al, Brain Research, 528, (1990), pp 48-54, describe the treatment of an in vitro amphibian neuronal preparation with Quin-2, membrane permeant calcium buffer, used to indicate the presence of calcium ions by fluorescence, for the purposes of determining the effect of this buffer upon intracellular calcium concentration, and resistance to excessive electrical stimulation under the application of the neurotoxin L-glutamate-sodium and the calcium ionophore A23187, a compound which makes the cell membrane permeable to the calcium ion.
Scharfman and Schwartzkroin, Science, 246, Oct. 13 (1989), pp 257-260, describe experiments in vitro that demonstrate that single neurons that have calcium binding proteins were more resistant to excessive electrical stimulation. Neurons with less calcium binding proteins were less resistant to excessive stimulation. Neurons with no calcium binding proteins into which a salt of a Ca.sup.2+ buffer was injected by micro-pipette became more resistant to excessive electrical stimulation than neurons into which the Ca.sup.2+ buffer was not injected. The authors concluded that effective buffering of intracellular calcium during periods of excessive excitation is crucial to neuronal survival. A further conclusion was that supplementing the calcium binding capacity of vulnerable neurons may prevent cell damage.
Billman G E, McIlroy B, Johnson J D (1991),"Elevated myocardial calcium and its role in sudden cardiac death. " FASEB J 5: 2586-2592 describes the treatment of cardiac arrhythmias with membrane permeant calcium chelators by the administration of a Ca.sup.2+ buffer to dogs. The dogs were found to have a lesser chance of having a fatal electrical dysfunction of the heart. This article teaches that when membrane permeant calcium buffers are given to dogs, the electrical activity of their hearts is altered.
Niesen C, Charlton M P, Carlen P L (1991) "Postsynaptic and presynaptic effects of the calcium chelator BAPTA on synaptic transmission in rat hippocampal dentate granule neurons". Brain Res 555: 319-325, shows that the membrane-permeant Ca.sup.2+ chelator BAPTA-AM can effect electrical activity of neurons when applied in vitro to an isolated brain slice preparation. The observed effects are similar to those seen when BAPTA salt is injected directly into neurons through a glass microelectrode. However, this article does not provide data to indicate that BAPTA-AM might be neuroprotective.
Carpenter-Deyo L, Duimstra J R, Hedstrom O, Reed D J (1991), "Toxicity to isolated hepatocytes caused by the intracellular calcium indicator, Quin 2". J Pharmacol Exp Therapeut 258: 739-746, teaches that membrane-permeant calcium buffers, (acetoxymethyl esters of Quin 2, Indo 1, Fluo 3, 5,5'-Dimethyl BAPTA) when applied to isolated liver cells, in vitro, cause toxicity to those cells. This article leads away from a teaching that membrane-permeant Ca.sup.2+ buffers prevent toxicity in vivo.
K. G. Baimbridge and K. M. Abdel-Hamid, "Intra-neuronal Ca.sup.2+ buffering with BAPTA enhances glutamate excitotoxicity in vitro and ischemic damage in vivo, "Society for Neuroscience Abstracts, 18, 1992, .571.4, 22nd Annual Meeting, Anaheim, Calif., Oct. 25-30, 1992, teaches that when BAPTA-AM is given to cultured neurons in vitro, the toxicity of glutamate is greatly enhanced. Further, that when BAPTA-AM is injected directly into the rat brain in vivo prior to giving the rat a stroke, the damaging effects of the stroke are greatly enhanced. This article also leads away from a teaching that membrane permeant Ca.sup.2+ buffers prevent in vivo toxicity.